UVM Theses and Dissertations
Format:
Print
Author:
Jennings, Mark E.
Dept./Program:
Chemistry
Year:
2007
Degree:
Ph. D.
Abstract:
Stable isotopically labeled materials are commonly used as tracers to determine in vivo lunetics. Because the amount of administered labeled material needs to be known to accurately measure tracer enrichments and calculate parameters describing human metabolism, the isotopic and chemical purities of the labeled material must be determined. This dissertation describes a method to determine the isotopic purity of the tracer, describes and compares two methods that determine the chemical purity of the tracer, and answers the question as to the significance of these methods relative to the calculations used to describe human metabolism.
Measurement of the distribution of isotopomers in a labeled compound (i.e. unlabeled, partially and fully labeled forms of the compound) defines the isotopic purity of the tracer. A method is presented that uses the measured mass spectral data of the unlabeled material to represent any and all combinations of isotopomer variations of that daterial and to determine abundances of these isotopomers. To demonstrate this method, the isotopomer distributions of samples of ¹³C-labeled leucine and glucose for both highly enriched isotopomers and labeled isotopomers present in low abundance against a natural isotopic abundance background were determined. The method accurately and precisely determined isotopomer identity and abundance in the labeled materials without adding noise or error that was not inherent in the original mass spectral data. In examples shown here, isotopomer uncertainties were calculated with relative standard errors of <1% from good quality mass spectral data.
The chemical purity methods described here define how much of the material is the labeled compound and its isotopomers relative to other chemicals such as salt or degradation products that may be present in the tracer material. Two methods are presented that use the measured mass spectral data to determine the chemical purity. The first method, the "intermediate standard" method, requires measuring the base masses of the unlabeled material, the labeled material, and a third material used as an intermediate standard that is either a highly labeled form of the labeled material or a chemical analogue. The second method, the "direct mixture" method, requires measuring the envelope of isotope ions around the base mass of the unlabeled material, the labeled material, and a mixture of the two.
To demonstrate these methods, the chemical purities of samples of ¹⁵N-labeled glycine, ¹³C₂-labeled glutamate, and ²H₃-labeled leucine were determined. Although both methods have the ability to accurately and precisely determine the chemical purity in the- labeled materials, the direct mixture method handles isotope effects caused by derivatization of the materials and ionization effects caused by the mass spectrometer. Hence the direct mixture method is more robust in determining the chemical purity of the labeled materials. In examples shown here, chemical purity uncertainties were calculated with relative standard errors of <2% from good quality mass spectral data.
The impact of including the measured chemical and isotopic purities versus assuming these purities were 100% was determined for the tracer enrichments and the calculated metabolism values. If the impact was not significant, then critical values were determined using the t-distribution, and the combined chemical and isotopic purity, or "overall purity," necessary to achieve the critical values was calculated. In general, an overall purity below 97% can produce significant differences in the enrichment measurements, and an overall purity below 94% can produce significant differences in calculations used to describe human metabolism.
Measurement of the distribution of isotopomers in a labeled compound (i.e. unlabeled, partially and fully labeled forms of the compound) defines the isotopic purity of the tracer. A method is presented that uses the measured mass spectral data of the unlabeled material to represent any and all combinations of isotopomer variations of that daterial and to determine abundances of these isotopomers. To demonstrate this method, the isotopomer distributions of samples of ¹³C-labeled leucine and glucose for both highly enriched isotopomers and labeled isotopomers present in low abundance against a natural isotopic abundance background were determined. The method accurately and precisely determined isotopomer identity and abundance in the labeled materials without adding noise or error that was not inherent in the original mass spectral data. In examples shown here, isotopomer uncertainties were calculated with relative standard errors of <1% from good quality mass spectral data.
The chemical purity methods described here define how much of the material is the labeled compound and its isotopomers relative to other chemicals such as salt or degradation products that may be present in the tracer material. Two methods are presented that use the measured mass spectral data to determine the chemical purity. The first method, the "intermediate standard" method, requires measuring the base masses of the unlabeled material, the labeled material, and a third material used as an intermediate standard that is either a highly labeled form of the labeled material or a chemical analogue. The second method, the "direct mixture" method, requires measuring the envelope of isotope ions around the base mass of the unlabeled material, the labeled material, and a mixture of the two.
To demonstrate these methods, the chemical purities of samples of ¹⁵N-labeled glycine, ¹³C₂-labeled glutamate, and ²H₃-labeled leucine were determined. Although both methods have the ability to accurately and precisely determine the chemical purity in the- labeled materials, the direct mixture method handles isotope effects caused by derivatization of the materials and ionization effects caused by the mass spectrometer. Hence the direct mixture method is more robust in determining the chemical purity of the labeled materials. In examples shown here, chemical purity uncertainties were calculated with relative standard errors of <2% from good quality mass spectral data.
The impact of including the measured chemical and isotopic purities versus assuming these purities were 100% was determined for the tracer enrichments and the calculated metabolism values. If the impact was not significant, then critical values were determined using the t-distribution, and the combined chemical and isotopic purity, or "overall purity," necessary to achieve the critical values was calculated. In general, an overall purity below 97% can produce significant differences in the enrichment measurements, and an overall purity below 94% can produce significant differences in calculations used to describe human metabolism.